Biochemistry 1993,32, 2003-2012
2003
Structural Studies of the Lipooligosaccharides from Haemophilus influenzae Type b Strain A2t Nancy J. Phillips,$ Michael A. Apicella,* J. McLeod Griffiss,ll and Bradford W. Gibson'.* Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, Department of Medicine, State University of New York, Buffalo, New York 14215, and Centrefor Immunochemistry, Department of Laboratory Medicine and Veterans Affairs Medical Center, University of California, San Francisco, California 94143 Received October 22. 1992
The outer membrane lipooligosaccharides (LOS) from Haemophilus influenzae type b strain A2 are a heterogeneous mixture of glycolipids containing a conserved Lipid A structure and a variable oligosaccharide moiety. After 0-deacylation by treatment with anhydrous hydrazine, the 0-deacylated LOS mixture was analyzed by electrospray mass spectrometry and shown to contain 11 components, ranging in M,from 2277.8 to 3416.4. The majority of these structures contained a variable number of hexoses, three L-glycero-D-manno-heptoses,and one 3-deoxy-D-manno-octulosonic acid (KDO) residue attached to a diphosphorylated 0-deacylated Lipid A moiety. Additional phosphate and phosphoethanolamine (PEA) groups were also present on the oligosaccharide structures. Two minor high molecular weight components were also observed that contained N-acetylhexosamine and sialic acid. Neuraminidase treatment of the 0-deacylated LOS mixture resulted in the loss of sialic acid from these latter two species. After mild acid hydrolysis and separation by size-exclusionchromatography, liquid secondary ion mass spectrometry identified six major and four minor oligosaccharides, ranging in M, from 1243.4 to 2215.8. These released oligosaccharides contained a common heptose trisaccharide core structure with anhydro-KDO a t the reducing terminus, which arises as an artifact of the hydrolysis procedure by j3-elimination of a phosphate group from the 4-position of KDO. Selected oligosaccharide fractions were subjected to composition and methylation analyses and sequenced by tandem mass spectrometry. Taken together, these data defined the major 0-deacylated LOS as follows: ABSTRACT:
Glc 1+4Glc 1+4Hep 1+KDO+O-deacylated Lipid A 41 P03& 1 Glc 1-14Glc 1+3Hep-PEA
3
T1 HeP
Higher molecular weight structures in the mixture contained galactose, N-acetylglucosamine, and sialic acid as additional branch sugars, suggesting that H . influenzae A2 is capable of forming a sialylated lactosamine structure.
In developed countries, Haemophilus influenzae type b (Hib)' is the leading cause of bacterial meningitis in young children (Moxon, 1990). The major virulence factor of this Gram-negative pathogen is its capsular polysaccharide, poly(ribosy1)ribitol phosphate (Turk, 1982). Bacterial outer? Financial support was provided by grants from the National Institute of Allergy and Infectious Diseases (A121620 and AI24616). We would also like to acknowledge the Mass Spectrometry Facility (A. Burlingame, director) with support from the National Center for Research Resources (RR01640), the National Science Foundation Biological Instrumentation Program, and the Research Service of the Department of Veterans Affairs. This is Report 7 1 from the Centre for Immunochemistry of the University of California, San Francisco. * To whom correspondence should be addressed at the School of Pharmacy 9264, 513 Parnassus Ave., University of California, San Francisco, CA 94143-0446. Department of Pharmaceutical Chemistry, UCSF.
*
I SUNY. 11 Department of Laboratory Medicine and VA Medical Center, UCSF.
membrane components, however, also modulate the pathogenicity of Hib. Recent studies of isogenic type b strains have demonstrated that changes in lipooligosaccharide (LOS) expression can alter the virulence of the pathogen (Cope et al., 1990;Gilsdorf & Ferrieri, 1986;Kimura & Hansen, 1986). Wild-type Hib strains in particular feature a great degree of structural and antigenicvariabilityin their LOS (Inzana, 1983; Abbreviations: ESMS, electrospray mass spectrometry; Gal, galactose, GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, Nacetylglucosamine; Hep, L-glycero-D-munno-heptose;Hex, hexose; HexNAc, N-acetylhexosamine; Hib, Huemophilus influenzue type b; HPLC, high-performance liquid chromatography; KDO, 3-deoxy-~-munnooctulosonic acid; LOS, lipooligosaccharide; LSIMS, liquid secondary ion mass spectrometry; MAb, monoclonal antibody; (M - H)-, deprotonated molecular ion; MS/MS, tandem mass spectrometry; NANA, N-acetylneuraminic acid (sialic acid); PEA, phosphoethanolamine; PMAA, partially methylated alditol acetate; PPEA, pyrophosphoethanolamine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
0006-2960/93/0432-2003$04.00/0 0 1993 American Chemical Society
2004 Biochemistry, Vol. 32, No. 8, 1993 Patrick et al., 1989), with phase variation of LOS epitopes occurring at high frequency (Kimura & Hansen, 1986;Kimura et al., 1987;Weiser et al., 1989). Thesecharacteristics suggest that variation of LOS surface structures may be an important mechanism for Hib evasion of host defense mechanisms. Like neisserial LOS, the LOS of H. influenzae consist of an oligosaccharideketosidicallylinked via one or more 3-deoxyD-manno-octulosonicacid (KDO) residues to a Lipid A moiety. Previous compositional studies have shown that H. influenzae LOS contain glucose, galactose, and L-glycero-D-mannoheptose as common sugar constituents, in addition to glucosamine and galactosamine in some strains (Flesher & Insel, 1978; Inzana et al., 1985; Parr & Bryan, 1984; van Alphen et al., 1990; Zamze & Moxon, 1987). The KDO residue present in H. influenzae LOS has been found to be phosphorylated in both a recombinant (Helander et al., 1988) and a nontypable strain (Phillips et al., 1992). Structural heterogeneity observed in H. influenzae LOS has been associated with the oligosaccharide moieties (Campagnari et al., 1987; Patrick et al., 1987), which contain both strain specific and conserved epitopes. LOS from Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus ducreyi have been found to share common epitopes with H. influenzae LOS, and recent studies have shown that some of these shared epitopes resemble host carbohydrate antigens (Campagnari et al., 1990; Mandrell et al., 1988; Virji et al., 1990) and are partially sialylated (Mandrell et al., 1991, 1992). In this report, we describe the preliminary structural characterization of the LOS from Hib strain A2. DNA from this Hib strain was recently used to construct a genomic library in the X bacteriophage EMBL3 (Spinola et al., 1990). Clones produced in this study expressed an Hib LOS oligosaccharide epitope in Escherichia coli. One clone, designated EMBLOS1, appeared to produce a LOS with a 1.4-kDa oligosaccharide added to the 4.1-kDa lipopolysaccharide of the E. coli strain which was transfected. Monoclonal antibody (MAb) 6E4, which recognizes a stable epitope on two components in the Hib A2 LOS mixture, also recognized the novel 5.5-kDa component in the chimeric LOS. The chemical structure of this Hib epitope has not yet been elucidated, although a hexasaccharide from a nontypable strain of H. influenzae which also expresses the epitope was recently described (Phillipset al., 1992). ThepreliminaryHibA2LOSstructures described here share common features with the LOS from the nontypable strain but contain more highly branched carbohydrate structures. Genetic studies have suggested that the Hib A2 LOS may express the 6E4 epitope on a separate oligosaccharide branch from the phase-varying epitopes (McLaughlin et al., 1992). The partial LOS structures described here provide a structural basis for this hypothesis. EXPERIMENTAL PROCEDURES Materials LPS from Salmonella typhimurium T V l l 9 Ra mutant, galactose, glucose, galactosamine, glucosamine, 3-deoxy-~manno-octulosonic acid (KDO), and anhydrous hydrazine were all obtained from Sigma (St. Louis, MO). Aqueous H F (48%) was purchased from Mallinckrodt (Muskegon, MI). Water (18 M a ) required for the Dionex chromatography was generated from deionized water using a Millipore Milli-Q water purification system. All other reagents and solvents used were of reagent grade.
Phillips et al. Methods Isolation of LOSfrom Hib Strain A2. Hib strain A2 was isolated from the cerebrospinal fluid of an Alaskan child as previously described (Spinola et al., 1990). LOS from this Hib strain was prepared by the extraction procedure of Darveau and Hancock (1983). 0-Deacylation of LOS. Hib A2 LOS (1.7 mg) was 0-deacylated by treatment with anhydrous hydrazine following the procedure of Helander et al. (1988) as previously described (Phillips et al., 1992). Neuraminidase Treatment of 0-Deacylated LOS. A portion of the 0-deacylated A2 LOS sample ( ~ 5 pg) 0 was treated with 50 milliunits of neuraminidase (Sigma Type VI) in 50 pL of PBS, pH 6.0, for 2 h at 37 "C. The enzyme digest was desalted by microdialysis against H2O (Pierce, microdialyzer system 500) using a 1000 MW cutoff membrane (Spectra/Por). The retentate was lyophilized and later redissolved in H2O for mass spectral studies. Isolation and Purification of Oligosaccharide and Lipid A Fractions. The LOS from Hib strain A2 (14 mg) and commercially available Salmonella typhimurium TV 119 Ra mutant (20 mg) were hydrolyzed in 1% acetic acid (2 mg of LOS/mL) for 2 h at 100 "C. The hydrolysates were centrifuged at 5000gfor 20 min at 4 "C and the supernatants removed. Pellets were washed with 1-3 mL of H2O and centrifuged again (SOOOg, 20 min, 4 "C). The supernatants and washings were pooled and lyophilized to give the oligosaccharidefractions. The Lipid A pellets were partitioned in CHCl3/methanol/O.l N HCl (2/1/2 v/v/v), and the organic layers plus emulsion layers were combined and evaporated to dryness. The Hib A2 oligosaccharide fraction (7.0 mg) was dissolved in 0.3 mL of 0.05 M pyridinium acetate buffer (pH 5.2), centrifuge-filtered through a 0.45-pm Nylon-66 membrane (Microfilterfuge tube, Rainin), and applied to two Bio-Gel P-4 columns connected in series (1.6 X 79 cm and 1.6 X 76.5 cm,
